1. Field of the Invention
This invention relates to a method and a device for optically discriminating nucleic acid bases (e.g., nucleotides) constituting a gene and for determining a sequence of the nucleic acid bases. The present invention specifically relates to an art in which fluorescence or luminescence of fluorescent molecules represented by the bases is utilized to discriminate the number, position, etc. of fluorescent molecules.
2. Related Art
DNA (deoxyribonucleic acid containing a base as a main component, with sugar and phosphoric acid bonded to the base), which is a composition material of a gene, exists as a strand of base pairs in a double helical structure. The double helix contains genetic information in code-like form (i.e., a base sequence). Genes are gathered in strings in the cell nucleus. Lower organisms, such as microorganisms, have thousands of nucleotide pairs at most, but higher organisms having more genetic information have several billion to an estimated 29 billion base pairs.
Genetic information is determined by a sequence of four kinds of bases--adenine (A), guanine (G), cytosine (C), and thymine (T). Accordingly, it is of great significance to know the base sequences for the future development of such fields as genetic engineering, medicine, etc.
It is known that these bases emit an intrinsic fluorescence, which increases at low temperatures (e.g., 100 K or less). It is possible to discriminate the bases in principle by examining their fluorescent lifetimes. To generate fluorescence, it is necessary to irradiate the bases with excitation light. A high sensitivity detector, such as a photomultiplier, is suitable for detecting fluorescence. The apparatus disclosed in, e.g., "Proc. Natl. Acad. Sci.", U.S.A., 86 (1989) 4087-91 (a first conventional method) is known for detecting a single fluorescent molecule. In this reference, as shown in FIG. 1, excitation light (laser beam) from a light source is applied to a flow cell 61 containing dye solution. Fluorescence is detected by a photomultiplier 63 in a direction which is normal to both the direction of irradiation of the excitation light and the direction of flow of the dye solution.
In FIG. 1, an optical system for forming an image on the photomultiplier 63 comprises a lens 64, an aperture 65, a wavelength selecting filter 66, and a condensation lens 67. A measuring system for measuring the fluorescence detected by the photomultiplier 63 comprises an electric signal detecting/multiplying unit 68, a fluorescent photon counter 69, and a computer 70.
A second conventional method in which respective single-fragment bases are modified by fixable fluorescent dyes is disclosed in Japanese Patent Laid-Open Publication No. 100945/1991, U.S. Pat. No. 4,962,037. This second conventional method has the same arrangement in which the respective bases are labelled by their characteristic dyes and then cut off by exonuclease III. A sequence of the bases is determined based on difference in fluorescence spectrum.
A third conventional method for single fluorescent molecule detection relies on high resolution spectroscopy of a single impurity aromatic molecule (pentacene) embedded in an organic molecule (paraterphenyl). This third conventional method, which is disclosed in J. Chem. Phys. 95(10), 15 Nov. 1991, 7150-7163, is not suitable for detecting the base. However, this method measures a fluorescence excitation spectrum of pentacene at ultra-low temperature (about 4K) to measure a uniformly wide spectrum in a non-uniformity wide spectrum, thereby using the former as a fluorescence spectrum of the single molecule.
On the other hand, advantages of the first conventional method produced by using a flow cell 61 are that degradation of dyes can be suppressed and that a filter can be provided in a dye circulation system to remove dust. But fluorescence can be observed only in the limited period of time (about 1 .mu.sec) in which the molecule is passing through a region irradiated with the excitation light. Accordingly, in the device of FIG. 1, the fluorescence from the bases of genes cannot be correctly and efficiently detected. Although only four kinds of bases (A, G, C, and T) are contained in DNA, a large quantity of each kind of base is present. However, because their sizes are very small, fluorescence generated from these bases is very feeble. Consequently, it is very difficult to discriminate type of bases solely on the basis of the short fluorescence observation period.
A longer period of fluorescence emission and higher efficiency can be obtained by irradiating the excitation light to a base flow over a larger area in the direction of the base flow. But in this case, the bases in the base flow are simultaneously detected, making the processing of the obtained data difficult.